Amino acid dysiherbaine

ABSTRACT

The present invention provides a novel amino acid dysiherbaine which is a non-NMDA type glutamate receptor agonist as well as salts and biological precursors thereof, which are useful as experimental materials for elucidating neuronal death or the functions of signal transduction of the central nervous system associated with glutamate receptors and which provide a possibility for the development of a glutamate receptor blocker. 
     A novel amino acid dysiherbaine represented by the formula (1): ##STR1## is obtained by purifying an aqueous extract of a sponge such as D. herbacea on the basis of toxicity to mice by liquid chromatography using Sephadex LH20 or the like and HPLC using a C18 column, and optionally converting it into a biological precursor thereof.

FIELD OF THE INVENTION

The present invention relates to a novel glutamate receptor agonist,more specifically a novel amino acid dysiherbaine which is an agonistfor non-NMDA type glutamate receptors as well as salts and biologicalprecursors thereof.

PRIOR ART

L-glutamic acid has attracted attention as an excitatoryneurotransmitter in central nervous systems of mammals, aneuroexcitotoxin which destroys neurocytes to cause variouscerebro-neuropathies, and a substance which has an important role in theformation of memory or learning.

L-glutamate receptors are associated with such a variety ofphysiological functions and classified into two types, i.e. ionotropicreceptors (iGluR) and metabotropic receptors (mGluR). Ionotropicreceptors are subclassified based on the recent information on exogenousagonists into the following three subtypes:

(a) NMDA type,

(b) KA type, and

(c) AMPA type

wherein NMDA=N-methyl-D-aspartic acid, KA=kainic acid,AMPA=α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and theseabbreviations will be hereinafter sometimes used.

KA type and AMPA type are sometimes collectively called as non-NMDAtype.

Among non-NMDA type receptors, KA type receptors are thought to beinvolved in neuronal death since it has been found that kainic aciditself induces epileptoid lesions and neuronal degeneration in rats. Onthe other hand, AMPA receptors are considered to be involved in normalrapid transmission in central nervous systems and the activation of AMPAreceptors seem to trigger the activation of NMDA type receptors. Inrecent years, researches on the level of molecular biology have showedthat ionotropic L-glutamate receptors types 1-4 (iGluR 1-4) correspondto receptors of AMPA type while types 5-7 (iGluR 5-7) and types KA1, 2correspond to receptors of KA type.

SUMMARY OF THE INVENTION

As described above, stimulation of glutamate receptors by L-glutamicacid plays an important role in living bodies, including neuronal deathand the signal transduction of the central nervous system. Thus,glutamate receptor agonists are experimental materials indispensable forexplaining the functions of the nervous system. However, the only knownagonists for non-NMDA type (particularly KA type) receptors are kainoids(collective name of the compounds which have a kainic acid skeleton intheir molecule) such as naturally occurring domoic acid and acromelicacid. Therefore, there is a demand for development ofagonists/antagonists with a new skeleton for use in investigations ofthe functions of the receptors.

DETAILED DESCRIPTION OF THE INVENTION

Bearing in mind that a number of natural marine products known aspoisons can act at micro-doses on systems which are involved inintracellular/intercellular signal transduction, we have made an attemptto isolate a novel compound with a specific physiological activity. As aresult, we found that an aqueous extract of a species of Micronesiansponge (Dysidea herbacea) induces characteristic conditions such asparoxysmal spasm in mice.

As a result of careful studies to isolate and purify the activesubstance inducing these conditions on the basis of the above finding,we have succeeded in isolating a novel amino acid represented by thefollowing chemical formula (1): ##STR2## from an aqueous extract of D.herbacea on the basis of toxicity to mice by combining liquidchromatography and high performance liquid chromatography (HPLC). Thecompound of this structure is an agonist for non-NMDA type glutamatereceptors, and named dysiherbaine.

The compound of the chemical formula (1) in the form of a salt or abiological precursor in which one or each of two carboxyl groups forms alower alkyl ester, for example, will be converted in vivo intodysiherbaine and also shows its biological activity.

Accordingly, the present invention provides a novel amino aciddysiherbaine as well as salts and biological precursors thereof asagonists for non-NMDA type glutamate receptors.

Examples of lower alkyl esters of the compound of the present inventioninclude esters with a straight or branched lower alkyl group containing1 to 5 carbon atoms, such as methyl ester, ethyl ester, n- andiso-propyl ester, n-, sec- and tert-butyl ester, n-, sec- andtert-pentyl ester, which are obtained by treating dysiherbaine byconventional esterification procedures.

Examples of salts of the compound of the present invention include acidaddition salts, alkali metal salts, alkali earth metal salts, etc. whichare obtained from dysiherbaine by conventional procedures, and they arepreferably physiologically acceptable if used as pharmaceuticals orbiochemical reagents or the like. Such acid addition salts includelactate, acetate, succinate, maleate, fumarate, tartrate, citrate,gluconate, ascorbate, benzoate, methanesulfonate, cinnamate,benzenesulfonate, or phosphate, hydrogenphosphate, hydrochloride,hydrobromide, hydroiodide, sulfamate, sulfate and hydrogensulfate, etc.Such alkali metal salts include sodium salt and potassium salt, andalkali earth metal salts include calcium salt.

Compound (1) of the present invention is obtained by purifying anaqueous extract of a sponge containing said compound such as D. herbaceaon the basis of toxicity to mice by liquid chromatography using SephadexLH20 or the like and high performance liquid chromatography (HPLC) usinga C18 column until a UV absorption monitor at 210 nm shows a singlepeak.

When intraperitoneally administered to mice, Compound (1) of the presentinvention induces scratching on the flank at a low dose (20 μg/kg),epileptoid spasm at a moderate dose (1.3 mg/kg) and violent attackleading to death at a higher dose (6.5 mg/kg).

Compound (1) of the present invention inhibits [³ H] kainic acid and [³H] AMPA, but not an NMDA antagonist [³ H] CGS-19755(cis-4-phosphonomethyl-2-piperidinecarboxylic acid) binding to synapticmembrane samples prepared from rat brain.

Moreover, Compound (1) induces a stronger inward current than kainicacid in primary cultures of rat cerebral cortex neurocytes. This currentinduction response is inhibited by a non-NMDA antagonist CNQX(6-cyano-7-nitroquinoxaline-2, 3-dione) but not an NMDA antagonist MK801((5R, 10S)-(+) 5-methyl-10,11-dihydro-5H-dibenzo [a, d] cycloheptene-5,10-imine). This means that Compound (1) is a non-NMDA type glutamateagonist acting on KA and AMPA type glutamate receptors.

Compound (1) of the present invention shows a stronger in vivo activitythan a known KA agonist domoic acid, so that it can be used as a usefulreagent of non-kainoid non-NMDA type glutamate agonist equally to kainicacid and domoic acid in the field of neuroscience, and promotesinvestigations of glutamate receptors including preparation of clinicalmodels to provide essential tools to the development of a glutamateblocker.

The following examples further explain the present invention in detailwithout, however, limiting the same thereto.

EXAMPLES Example 1

Isolation of Compound (1)

Step 1. Preparation of a crude extract from sponge

Sponges collected at a shoal (at depths of 3-5 m) of Yap, Micronesia inJuly 1995, D. herbacea (200 g), were homogenized with purified water (20ml), followed by centrifugation of 10,000 rpm at 4° C., to give anextract. To this extract was added 2-propanol (100 ml) to remove highmolecular substances by precipitation and the supernatant from thecentrifugation was collected, concentrated under reduced pressure andlyophilized to give a crude extract.

Step 2. Purification of Compound (1)

The obtained crude extract was dissolved in purified water (50 ml) andapplied to column chromatography on Sephadex LH20 (Pharmacia, 3.5×100cm) with purified water as an eluent. Fractions showing toxicity byintraperitoneal administration to mice were collected and applied toreverse phase column chromatography on ODS medium pressure column (WakoPure Chemicals, WAKO 60) with purified water as an eluent. Activefractions were collected and separated by column chromatography onSephadex LH20 (Pharmacia, 3.5×100 cm) again. Then, the active fractionswere passed at a flow rate of 1 ml/min through HPLC column of RP18(Bio-Rad, 1×25 cm) with purified water as a developing solvent. Peakfractions eluting at a retention time of about 14.6 minutes werecollected while monitoring UV absorption at 210 nm, thus 7 mg ofCompound (1) was obtained as a colorless amorphous product. Compound (1)gave a single peak at a retention time of 14.6 minutes during said HPLCand was named dysiherbaine.

Instrumental analyses:

TLC (C-18): Rf=0.7 (H₂ O), Rf=0.4 (methanol/0.5 M-NaCl=5:1).

[α]D=-3.5° (c 0.4, H₂ O, 26° C.).

CD (H₂ O): λext 223 nm, Δε-1.4; λext 203 nm, Δε11.6.

PMR (500 MHz, D₂ O, δppm): 1.93 (dd, 1H, J=11.5, 15.0 Hz), 2.15 (dd, 1H,J=3.5, 14.0 Hz), 2.58 (dd, 1H, J=0.5, 14.0 Hz), 2.59 (dd, 1H, J=2.5,15.0 Hz), 2.75 (s, 1H), 3.47 (dd, 1H, J=2.5, 11.5 Hz), 3.54 (dd, 1H,J=1.0, 13.0 Hz), 3.55 (dd, 1H, J=3.5, 3.5 Hz), 3.85 (m, 1H), 3.88 (dd,1H, J=2.5, 13.0 Hz), 4.15 (brs, 1H), 4.30 (m, 1H).

ESIMS: m/Z 305 ( +H), 327 (M+Na).

HRFABMS: calcd for C₁₂ H₂₁ N₂ O₇ 305.1349, found 305.1348.

These data revealed that dysiherbaine is represented by the abovechemical formula (1) and an estimated stereoisomeric form of thechemical formula (la): ##STR3##

Example 2

Synthesis of dysiherbaine dimethyl ester

To a solution of dysiherbaine obtained in Example 1 (2.2 mg) in methanol(0.9 ml) was added an ice-cooled 10% thionyl chloride/methanol solution(100 μl) and the mixture was stirred at room temperature for 24 hours.Methanol was distilled off and the residue was dissolved in water (1ml), then lyophilized to afford the title compound in a quantitativeyield.

Instrumental analyses:

TLC (C-18): Rf=0.7 (methanol/0.5 M-NaCl=5:1).

PMR (500 MHz, CD₃ OD, δppm): 2.25-2.35 (m, 1H), 2.37 (m, 1H), 2.73 (d,1H, J=13.5 Hz), 2.82 (m, 1H), 2.87 (s, 1H), 3.62 (d, 1H, J=13 Hz), 3.65(m, 1H), 3.85-3.88 (m, 6H), 3.89 (brs, 1H), 3.95 (dd, 1H, J=5, 13 Hz),4.07 (m, 1H), 4.22 (brs, 1H), 4.55 (m, 1H).

Evaluation Example 1

Intraperitoneal administration to mice

Compound (1) was diluted with purified water and intraperitoneallyinjected into a DDY mouse (male, 15 g). At a low dose (20 μg/kg), theanimal was found to repeat scratching on the flank for 10-20 minutes. Ata moderate dose (1.3 mg/kg), the animal showed epileptoid spasm andrecovered from this condition on the following day. At a higher dose(6.5 mg/kg), however, the animal died in about 40 minutes after violentattack.

Evaluation Example 2

Radiobinding assay in rat brain synaptic membrane samples

Rat brain synaptic membrane samples were prepared according to thedescribed protocols (London, E. D., Coyle, J. T., Mol. Pharmacol. 1979,15, 492; Murphy, D. E., Snowhill, E. W.; Williams, M., Neurochem. Res.1987, 12, 775 and Murphy, D. E., Hutchison, A. J., Hurt, S. D.,Williams, M., Sills, M. A., Br. J. Pharmacol. 1988, 95, 932).Radioligands and incubation conditions used in the binding assay as wellas experimental results are as follows:

KA receptors

[Conditions]: ligand [³ H] kainic acid 1 nM; incubation medium 100 mMTris-acetate buffer (pH 7.1); incubation condition 4° C., 1 hour.

[Results]: Compound (1) concentration-dependently inhibited [³ H] kainicacid binding to KA receptors. IC₅₀ values of Compound (1), glutamic acidand kainic acid are shown below.

    ______________________________________                                        Compound (1)   59 ± 7.8   nM                                                 Glutamic acid 110 ± 18 nM                                                  Kainic acid 4.3 ± 0.5 nM                                                 ______________________________________                                    

AMPA receptors

[Conditions]: ligand [³ H] AMPA 5 nM; incubation medium 100 mM KSCN/50mM Tris-acetate buffer (pH 7.4); incubation condition 4° C., 1 hour.

[Results]: Compound (1) also concentration-dependently inhibited [³ H]AMPA binding to AMPA receptors. IC₅₀ values of Compound (1), glutamicacid and AMPA are shown below.

    ______________________________________                                        Compound (1)   224 ± 22   nM                                                 Glutamic acid 124 ± 41 nM                                                  AMPA 5.6 ± 1.1 nM                                                        ______________________________________                                    

NMDA receptors

[Conditions]: ligand [³ H] CGS-19755 10 nM; incubation medium 50 mMTris-acetate buffer (pH 8.0); incubation condition 4° C., 1 hour.

[Results]: Compound (1) did not inhibit [³ H] CGS-19755 binding to NMDAreceptors under these conditions.

Evaluation Example 3

Electrophysiological evaluation in primary cultures of rat cerebralcortex neurocytes

Glutamate receptors expressed in primary cultures of rat cerebral cortexneurocytes grown for 10-21 days were observed for their receptorresponse to agonists by an electrophysiological method (patch clampwhole cell method) (Murase, K., Randic, M., Shirasaki, T., Nakagawa, T.,Akaike, N., Brain Res. 1990, 525, 84). Compound (1)concentration-dependently induced a strong inward current.

EC₅₀ values of Compound (1), kainic acid and AMPA are shown below.

Compound (1) EC₅₀ =4 μM

Max response=1.40 nA

AMPA EC₅₀ =4 μM

Max response=1.26 nA

Kainic acid EC₅₀ =40 μM

Max response=1.31 nA

The current induced by Compound (1) was significantly inhibited by CNQXbut not by MK801.

These evaluation examples revealed that Compound (1) is a non-NMDA typeglutamate receptor agonist.

According to the present invention, a novel amino acid dysiherbainewhich is a non-NMDA type glutamate receptor agonist is provided as wellas lower alkyl esters and salts thereof. This compound is useful forinvestigations of glutamate receptors including preparation of clinicalmodels to provide an essential tool for to the development of aglutamate receptor blocker.

The compound of the present invention also promotes investigations ofnon-NMDA type glutamate receptor agonists to provide a possibility tothe development a glutamate receptor blocker which is potentially usefulfor the therapy of neuropathies such as epilepsy, Huntington's diseaseand Parkinson's disease or neurodegenerations.

What is claimed is:
 1. A novel amino acid dysiherbaine represented bythe chemical formula (1): ##STR4## as well as salts and biologicalprecursors thereof.
 2. The compound of claim 1 represented by thechemical formula (1a): ##STR5## as well as salts and biologicalprecursors thereof.
 3. An agonist for non-NMDA type glutamate receptorscomprising the compound of claim 1 or 2 as an active ingredient.